Cycloalkyl-substituted glutaramide diuretic agents

ABSTRACT

2-Acylaminoalkyl-3-[1-(carboxycycloalkylcarbamoyl)cycloalkyl]propanoic acid derivatives (which may also be viewed as glutaramide derivatives) are useful in the treatment of hypertension, heart failure and renal insufficiency, based upon their inhibition of zinc-dependant, neutral endopiptidase.

BACKGROUND OF THE INVENTION

This invention relates to a series of cycloalkyl-substituted glutaramidederivatives which are diuretic agents having utility in a variety oftherapeutic areas including the treatment of various cardiovasculardisorders such as hypertension, heart failure and renal insufficiency.

The compounds are inhibitors of the zinc-dependent, neutralendopeptidase E.C.3.4 24 11. This enzyme is involved in the breakdown ofseveral peptide hormones, including atrial natriuretic factor (ANF),which is secreted by the heart and which has potent vasodilatory,diuretic and natriuretic activity. Thus, the compounds of the invention,by inhibiting the neutral endopeptidase E.C.3.4.24 11, can potentiatethe biological effects of ANF, and in particular the compounds arediuretic agents having utility in the treatment of a number ofdisorders, including hypertension, heart failure, angina, renalinsufficiency, premenstrual syndrome, cyclical oedema, Menieres disease,hyperaldosteronism (primary and secondary) pulmonary oedema, ascites,and hypercalciuria. In addition, because of their ability to potentiatethe effects of ANF the compounds have utility in the treatment ofglaucoma. As a further result of their ability to inhibit the neutralendopeptidase E.C.3.4.24.11 the compounds of the invention may haveactivity in other therapeutic areas including for example the treatmentof asthma, inflammation, pain, epilepsy, affective disorders, dementiaand geriatric confusion, obesity and gastrointestinal disorders(especially diarrhoea and irritable bowel syndrome), the modulation ofgastric acid secretion and the treatment of hyperreninaemia andleukemia.

Published European Patent application No. 0274234 of the same assigneediscloses structurally related compounds having the same utility.

SUMMARY OF THE INVENTION

The compounds are of the formula: ##STR1## wherein A completes a 4 to 7membered carbocyclic ring which may be saturated or mono-unsaturated andwhich may optionally be fused to a further saturated or unsaturated 5 or6 membered carbocyclic ring;

B is (CH₂)_(m) wherein m is an integer of from 1 to 3; each of R and R⁴is independently H, C₁ -C₆ alkyl, benzyl or an alternative biolabileester-forming group;

R¹ is H or C₁ -C₄ alkyl;

R² and R³ are each independently H, OH, C₁ -C₆ alkyl or C₁ -C₆ alkoxy;or R² and R³ are linked together and are (CH₂)_(r) wherein r is aninteger of from 1 to 4;

Y is an optional alkylene group of from 1 to 6 carbon atoms which may bestraight or branched-chain;

and

R⁵ is R⁶ CONR⁹ --, R⁶ SO₂ NR⁹ --, R⁶ CO₂ --, R⁶ CO--, R⁶ SO_(q) --, R⁷NR⁹ CO--, R⁷ NR⁹ SO₂ -- or R⁷ OCO--;

wherein

R⁶ is a group of the formula: ##STR2## R⁷ is a group of the formula:##STR3## and R⁹ is H, C₁ -C₆ alkyl, aryl, C₃ -C₇ cycloalkyl,heterocyclyl, aryl (C₁ -C₆ alkyl) or heterocyclyl(C₁ -C₆ alkyl);

wherein

R⁸ is R⁹ CONR⁹ --, R⁹ SO₂ NR⁹ --, R¹³ R¹⁴ N--(CH₂)_(p) --, or R⁹ O--,wherein each R⁹ is as previously defined above; R¹⁰ and R¹¹ are eachindependently H or C₁ -C₆ alkyl; or R¹⁰ is H and R¹¹ is C₁ -C₆ alkylwhich is substituted by OH, SH, SCH₃, NH₂, aryl(C₁ -C₆ alkyl)OCONH--,NH₂ CO--, CO₂ H, guanidino, aryl, or heterocyclyl; or the two groups R¹⁰and R¹¹ are joined together to form, with the carbon atom to which theyare attached, a 5 or 6 membered carbocyclic ring which may be saturatedor mono-unsaturated and which may optionally be substituted by C₁ -C₄alkyl or fused to a further 5 or 6 membered saturated or unsaturatedcarbocyclic ring;

or

R¹⁰ is H, n is 0 and R⁸ and R¹¹ are linked to form a 2-(N--COR⁹-4-aminopyrrolidinyl) group;

R¹² is R¹³ R¹⁴ NCO--, R⁹ OCO--, R⁹ OCH₂ -- or heterocyclyl, wherein R⁹is as previously defined above;

R¹³ and R¹⁴ are each independently H, C₁ -C₆ alkyl, C₃ -C₇ cycloalkyl,aryl, aryl(C₁ -C₆ alkyl), C₂ -C₆ alkoxyalkyl, amino (C₁ -C₆ alkyl),heterocyclyl or heterocyclyl(C₁ -C₆ alkyl); or the two groups R¹³ andR¹⁴ are taken together to form, with the nitrogen to which they areattached, a pyrrolidinyl, piperidino, morpholino, piperazinyl, N-(C₁ -C₄alkyl)piperazinyl, pyrrolyl, imidazolyl, pyrazolyl or triazolyl group;

n is 0 or 1;

p is 0 or an integer of from 1 to 6;

and

q is 0, 1 or 2;

and pharmaceutically acceptable salts thereof and bioprecursorstherefor.

In the above definition, unless otherwise indicated, alkyl groups havingthree or more carbon atoms may be straight or branched-chain. The termaryl as used herein means an aromatic hydrocarbon group such as phenyl,naphthyl or biphenyl which may optionally be substituted, for examplewith one or more OH, CN, CF₃, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, halo,carbamoyl, aminosulphonyl, amino, mono or di(C₁ -C₄ alkyl)amino or (C₁-C₄ alkanoyl)amino groups. Halo means fluoro, chloro, bromo or iodo.

The term heterocyclyl means a 5 or 6 membered nitrogen, oxygen orsulphur containing heterocyclic group which, unless otherwise stated,may be saturated or unsaturated and which may optionally include afurther oxygen or one to three nitrogen atoms in the ring and which mayoptionally be benzofused or substituted with for example, one or morehalo, C₁ -C₄ alkyl, hydroxy, carbamoyl, benzyl, oxo, amino or mono ordi-(C₁ -C₄ alkyl)amino or (C₁ -C₄ alkanoyl)amino groups. Particularexamples of heterocycles include pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,furanyl, tetrahydrofuranyl, tetrahydropyranyl, dioxanyl, thienyl,oxazolyl, isoxazolyl, thiazolyl, indolyl, isoindolinyl, quinolyl,quinoxalinyl, quinazolinyl and benzimidazolyl, each being optionallysubstituted as previously defined.

The compounds of formula (I) may contain several asymmetric centres andthus they can exist as enantiomers and diastereomers. The inventionincludes both mixtures and the separated individual isomers. Thesubstituents R², R³ and CO₂ R⁴ may have cis or trans geometry relativeto the amide attachment.

The pharmaceutically acceptable salts of the compounds of formula (I)containing an acidic centre are those formed with bases which formnon-toxic salts. Examples include the alkali metal salts such as thesodium, potassium or calcium salts or salts with amines such asdiethylamine. Compounds having a basic centre can also form acidaddition salts with pharmaceutically acceptable acids. Examples includethe hydrochloride hydrobromide, sulphate or bisulphate, phosphate orhydrogen phosphate, acetate, citrate, fumarate, gluconate, lactate,maleate, succinate and tartrate salts.

The term bioprecursor in the above definition means a pharmaceuticallyacceptable biologically degradable derivative of the compound of formula(I) which, upon administration to an animal or human being, is convertedin the body to produce a compound of the formula (I).

A preferred group of compounds of the formula (I) are those wherein A is(CH₂)₄, R¹ is H and B is (CH₂)₂, i.e. compounds of the formula (II)below wherein R, R²,R³, R⁴, Y and R⁵ are as previously defined forformula (I): ##STR4##

Also preferred are those compounds of formulae (I) and (II) wherein Rand R⁴ are both H (diacids) as well as biolabile mono and di-esterderivatives thereof wherein one or both of R and R⁴ is a biolabileester-forming group.

The term biolabile ester-forming group is well understood in the art asmeaning a group which provides an ester which can be readily cleaved inthe body to liberate the corresponding diacid of formula (I) wherein Rand R⁴ are both H. A number of such ester groups are well known, forexample in the penicillin area or in the case of the ACE-inhibitorantihypertensive agents.

In the case of the compounds of formulae (I) and (II) such biolabilepro-drug esters are particularly advantageous in providing compounds ofthe formula (I) suitable for oral administration. The suitability of anyparticular ester-forming group can be assessed by conventional animal orin vitro enzyme hydrolysis studies. Thus, desirably for optimum effect,the ester should only be hydrolysed after absorption, accordingly, theester should be resistant to hydrolysis before absorption by digestiveenzymes but should be readily hydrolyzed by for example, liver enzymes.In this way the active diacid is released into the bloodstream followingoral absorption.

In addition to lower alkyl esters (particularly ethyl) and benzylesters, suitable biolabile esters include alkanoyloxyalkyl esters,including alkyl, cycloalkyl and aryl substituted derivatives thereof,aryloxyalkyl esters, aroyloxyalkyl esters, aralkyloxyalkyl esters,arylesters, aralkylesters, and haloalkyl esters wherein said alkanoyl oralkyl groups have from 1 to 8 carbon atoms and are branched or straightchain and said aryl groups are phenyl, naphthyl or indanyl optionallysubstituted with one or more C₁ -C₄ alkyl or C₁ -C₄ alkoxy groups orhalo atoms.

Thus examples of R and R⁴ when they are biolabile ester-forming groupsother than ethyl and benzyl include: 1-(2,2-diethylbutyryloxy)ethyl,2-ethylpropionyloxymethyl, 1-(2-ethylpropionyloxy)ethyl,1-(2,4-dimethylbenzoyloxy)ethyl, α-benzoyloxybenzyl,1-(benzoyloxy)ethyl, 2-methyl-1-propionyloxypropyl,2,4,6-trimethylbenzoyloxymethyl, 1-(2,4,6-trimethylbenzoyloxy)ethyl,pivaloyloxymethyl, phenethyl, phenpropyl, 2,2,2-trifluoroethyl, 1- or2-naphthyl, 2,4-dimethylphenyl, 4-t-butylphenyl,5-(4-methyl-1,3-dioxalynyl-2-onyl)methyl and 5-indanyl.

Particularly preferred biolabile ester-forming groups are ethyl, benzyl,2,4-dimethylphenyl, 4-t-butylphenyl and 5-indanyl.

Compounds of the formulae (I) and (II) wherein one or both of R and R⁴are C₁ -C₆ alkyl, particularly ethyl, or benzyl, are also active byvirtue of their hydrolysis in vivo, and, in addition, are valuableintermediates for the preparation of the diacids wherein R and R⁴ areboth H.

In a further group of preferred compounds of formula (II), R, R² and R⁴are each H. R³ is preferably H or C⁴ -C⁶ alkyl especially n-butyl.Particularly preferred are those compounds wherein the carboxy groupCOO₂ R⁴ is attached at the 3- or 4-position of the cyclohexane ring,most especially those compounds having cis-stereochemistry relative tothe amide group.

In one aspect of the invention R⁵ is R⁶ CONR⁹, or R⁷ NR⁹ CO-- wherein R⁹is H and R⁶ is a group of the formula: ##STR5##

Particularly wherein R⁸ is (C₁ -C₆ alkyl)CONH--, arylCONH--, or (C₁ -C₆alkyl)SO₂ NH--, R¹⁰ is H and R¹¹ is C₁ -C₄ alkyl, benzyl or amino(C₁ -C₆alkyl).

In a further particular and preferred aspect of the invention Y ismethylene and R⁵ is N² -substituted-L-lysyl-amino, particularly wheresaid substituent is N² -acetyl, N² -benzoyl, N² -naphthoyl or N²-methanesulphonyl; thus preferred compounds are:

2-(N²-acetyl-L-lysylaminomethyl)-3-{1-[(cis-4-carboxycyclohexyl)carbamoyl]cyclopentyl}propanoicacid;

2-(N² -benzoyl-L-lysylaminomethyl)-3-{1-[cis-4-carboxy)cyclohexyl)carbamoyl]cyclopentyl}propanoic acid,

2-(N²-naphthoyl-L-lysylaminomethyl)-3-{1-[(cis-4-carboxycyclohexyl)carbamoyl]cyclopentyl}propanoicacid,

2-(N²-acetyl-L-lysylaminomethyl)-3-{1-[cis-4-carboxy-cis-3-butyl-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid,

2-(N²-acetyl-L-lysylaminomethyl)-3-{1-[cis-4-carboxytrans-3-butyl-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid,

2-(N²-methanesulphonyl-L-lysylaminomethyl)-3-{1-[cis-4-carboxy-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid,

2-(N²-methanesulphonyl-L-lysylaminomethyl)-3-{1[cis-4-carboxy-cis-3-(3-methylbutyl)cyclohexyl)carbamoyl)cyclopentyl}propanoicacid,

and 2-(N²-methanesulphonyl-L-lysylaminomethyl)-3-{1-[cis-4-carboxy-cis-3-butyl-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of formula (I) are prepared by a number of differentprocesses according to the invention:

(a) In one process, the compounds of formula (I) wherein R⁵ is R⁶ CONR⁹are prepared by a process which involves acylating an amine of theformula: ##STR6## wherein A, B, Y, R¹, R², R³ and R⁹ are as previouslydefined and R¹⁵ and R¹⁶ are as previously defined for R and R⁴ excludingH, or they are conventional carboxylic acid protecting groups; byreaction with an acid of the formula: ##STR7## wherein R⁶ is aspreviously defined, and wherein any reactive groups therein areoptionally protected, to yield a compound of the formula: ##STR8##wherein R^(6') is as previously defined for R⁶ with any reactive groupstherein optionally protected; and subsequently removing any protectinggroups, if present, and, if desired, hydrolysing the ester product toyield the carboxylic acids wherein R and R⁴ are H.

The reaction of the compounds of formula (III) and (IV) is achievedusing conventional amide coupling techniques. Thus in one process thereaction is achieved with the reactants dissolved in an organic solvent,e.g. dichloromethane, using a carbodiimide condensing agent, for example1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, orN,N'-dicyclohexylcarbodiimide, advantageously in the presence of1-hydroxybenzotriazole and an organic base such as N-methylmorpholine.The reaction is generally complete after a period of from 12 to 24 hoursat room temperature and the product is then isolated by conventionalprocedures, i.e. by washing with water or filtration to remove the ureabiproduct and evaporation of the solvent. The product may be furtherpurified by crystallisation or chromatography, if necessary. Thecompounds of formula (V) include compounds of formula (I) wherein R andR⁴ are C₁ -C₆ alkyl or benzyl.

The diesters of formula (V) may be further reacted to give the monoesteror diacid derivatives of formula (I) wherein one or both of R and R⁴ areH. The conditions used will depend on the precise nature of the groupsR¹⁵ and R¹⁶ present in the compound of formula (V) and a number ofvariations are possible. Thus for example when both of R¹⁵ and R¹⁶ arebenzyl, hydrogenation of the product will yield the diacid of formula(I) wherein R and R⁴ are both H. Alternatively if R¹⁵ is benzyl and R¹⁶is alkyl, hydrogenation will yield a monoester product. This can then behydrolysed, if desired, to again yield the diacid product. When one ofR¹⁵ and R¹⁶ is t-butyl, treatment of the compound of formula (V) withtrifluoroacetic acid yields the corresponding acid. The diester productwherein R¹⁵ and R¹⁶ are benzyl or lower alkyl can also be treated withtrimethylsilyl iodide to produce the dicarboxylic acid product. If someother carboxylic acid protecting group is used for R¹⁵ or R¹⁶ thenclearly appropriate conditions for its removal must be employed in thefinal step to give the ester or diacid product of formula (I). In thecase where the ring A or the substituent R⁵ is unsaturated, thedeprotection must be effected by non-reductive methods, thus for exampleif either of R and R⁴ is benzyl, they may be removed by treatment withtrimethylsilyl iodide.

Finally any protecting groups which may be present in R^(6') are removedby methods appropriate to the particular group used. Thus, for example,if an amino group is present in R⁶, this may be protected as thebenzyloxycarbonylamino group, the benzyloxycarbonyl group being removedin the final step by catalytic hydrogenation.

Compounds of the formula (I) where one or both of R and R⁴ are biolabileester forming groups are prepared following similar procedures, startingwith a compound of the formula (III) wherein R¹⁵ and/or R¹⁶ arebiolabile ester forming groups.

In each case the product may be obtained as the free carboxylic acid orit may be neutralised with an appropriate base and isolated in saltform.

The starting cycloalkyl-substituted glutaric acid mono esters of formulaIII may be prepared by a number of different processes as described inour European patent application no. 0274234.

The acids of formula IV are generally known compounds which are eithercommercially available or they may be prepared following literatureprecedents.

(b) In a further process, compounds of the formula (I) wherein R⁵ is R⁶SO₂ NR⁹ -- are prepared by an entirely analogous procedure by reactionof a sulphonyl halide of formula R⁶ SO₂ --hal with the amine of formula(III).

(c) Compounds of the formula (I) wherein R⁵ is R⁷ NR⁹ CO-- are preparedby reaction of a compound of the formula: ##STR9## wherein A, B, Y, R¹,R², R³, R¹⁵ and R¹⁶ are as previously defined, by reaction with an amineof the formula R⁷ R⁹ NH, followed by removal of any protecting groupswhich may be present and hydrolysis of the ester product to yield thecarboxylic acids wherein R and R⁴ are H.

The reaction of the compound of formula (IX) and the amine may beachieved using the amide coupling techniques already described underprocess (a) above. The subsequent steps are also as previouslydescribed. The compounds of formula (IX) are prepared following theprocedures described in our European patent application No. 0274234 toprovide the corresponding benzyl ester (where R⁵ is C₆ H₅ CH₂ CO₂ --),catalytic hydrogenation gives the carboxylic acid of formula (IX). Theamines of formula R⁷ R⁹ NH are generally derived from the naturallyoccurring amino acids with appropriate protection of reactive sidechains.

(d) In a further process compounds of formula (I) wherein R⁵ is R⁶ CONR⁹or wherein R⁵ is R⁶ CO₂ --, R⁶ CO--, R⁶ SO_(q) --, R⁷ NR⁹ SO₂ -- or R⁷OCO are prepared following the synthetic procedure described in ourEuropean patent application no. 0274234, i.e. using the followingprocess: ##STR10## wherein A, B, Y, R¹, R², R³, R¹⁵ and R¹⁶ are aspreviously defined and R^(5') is as defined by R⁵ with any reactivegroups therein optionally protected.

The protecting and coupling techniques required are as previouslydescribed. The compounds of formulae (VI) and (VII) may be preparedfollowing the general procedures described in the above-mentionedEuropean patent application.

As previously mentioned, the compounds of the invention are potentinhibitors of the neutral endopeptidase (E.C.3.4.24.11). This enzyme isinvolved in the breakdown of a number of peptide hormones and, inparticular it is involved in the breakdown of atrial natriuretic factor(ANF). This hormone consists of a family of related natriureticpeptides, secreted by the heart, of which the major circulating form inhumans is known to be the 28 amino-acid peptide referred to as α-hANP(see for example G. A. Sagnella and G. A. MacGreggor, Nature, 1984, 309,666 and S. A. Atlas and others, Nature, 1984, 309, 717-725). Thus, thecompounds of the invention, by preventing the degradation of ANF, byendopeptidase E.C.3.4.24.11 can potentiate its biological effects andthe compounds are thus diuretic and natriuretic agents of utility in anumber of disorders as previously described.

Activity against neutral endopeptidase E.C.3.4.24.11 is assessed using aprocedure based on the assay described by J. T. Gafford, R. A. Skidgel,E. G. Erdos and L. B. Hersh, Biochemistry, 1983, 32, 3265-3271. Themethod involves determining the concentration of compound required toreduce by 50% the rate of release of radiolabelled hippuric acid fromhippuryl-L-phenylalanyl-L-arginine by a neutral endopeptidasepreparation from rat kidney.

The activity of the compounds as diuretic agents is determined bymeasuring their ability to increase urine output and sodium ionexcretion in saline loaded conscious mice. In this test, male mice(Charles River CD1, 22-28 g) are acclimatised and starved overnight inmetabowls. The mice are dosed intravenously via the tail vein, with thetest compound dissolved in a volume of saline solution equivalent to2.5% of body weight. Urine samples are collected each hour for two hoursin pre-weighed tubes and analysed for electrolyte concentration. Urinevolume and sodium ion concentration from the test animals are comparedto a control group which received only saline.

For administration to man in the curative or prophylactic treatment ofhypertension, congestive heart failure or renal insufficiency, oraldosages of the compounds will generally be in the range of from 4-800 mgdaily for an average adult patient (70 kg). Thus for a typical adultpatient, individual tablets or capsules contain from 2 to 400 mg ofactive compound, in a suitable pharmaceutically acceptable vehicle orcarrier for administration singly, or in multiple doses, once or severaltimes a day. Dosages for intravenous administration would typically bewithin the range 1 to 400 mg per single dose as required. In practicethe physician will determine the actual dosage which will be mostsuitable for an individual patient and it will vary with the age, weightand response of the particular patient. The above dosages are exemplaryof the average case but there can, of course, be individual instanceswhere higher or lower dosage ranges are merited, and such are within thescope of this invention.

For human use, the compounds of the formula (I) can be administeredalone, but will generally be administered in admixture with apharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. For example, theymay be administered orally in the form of tablets containing suchexcipients as starch or lactose, or in capsules or ovules either aloneor in admixture with excipients, or in the form of elixirs orsuspensions containing flavouring or colouring agents. They may beinjected parenterally, for example, intravenously, intramuscularly orsubcutaneously. For parenteral administration, they are best used in theform of a sterile aqueous solution which may contain other substances,for example, enough salts or glucose to make the solution isotonic withblood.

The compounds may be administered alone but may also be administeredtogether with such other agents as the physician shall direct tooptimise control of blood pressure or to treat congestive heart failure,renal insufficiency or other disorders in any particular patient inaccordance with established medical practice. Thus the compounds can beco-administered with a variety of cardiovascular agents, for examplewith an ACE inhibitor such as captopril or enalapril to facilitate thecontrol of blood pressure in treatment of hypertension; or withdigitalis, or another cardiac stimulant or with an ACE inhibitor, forthe treatment of congestive heart failure. Other possibilities includeco-administration with a calcium antagonist (e.g. nifedipine, amlodipineor diltiazem) a beta-blocker (e.g. atenolol) or an alpha-blocker (e.g.prazosin or doxazosin) as shall be determined by the physician asappropriate for the treatment of the particular patient or conditioninvolved.

In addition to the above, the compounds may also be administered inconjunction with exogenous ANF, or a derivative thereof or relatedpeptide or peptide fragment having diuretic/natriuretic activity or withother ANF-gene related peptides (e.g. as described by D. L. Vesely etal, Biochem. Biophys. Res. Comm., 1987, 143, 186).

Thus in a further aspect the invention provides a pharmaceuticalcomposition comprising a compound of the formula (I), or apharmaceutically acceptable salt thereof or bioprecursor therefor,together with a pharmaceutically acceptable diluent or carrier.

The invention also includes a compounds of the formula (I), or apharmaceutically acceptable salt thereof or bioprecursor therefor, foruse in medicine, particularly for use as a diuretic agent for thetreatment of hypertension, congestive heart failure or renalinsufficiency in a human being.

The invention further includes the use of a compound of the formula (I)for the manufacture of a medicament for the treatment of hypertension,heart failure, angina, renal insufficiency, premenstrual syndrome,cyclical oedema, Menieres disease, hyperaldosteronism, pulmonary oedema,ascites, hypercalciuria, glaucoma, asthma, inflammation, pain, epilepsy,affective disorders, dementia and geriatric confusion, obesity,gastrointestinal disorders (including diarrhoea), hyperreninaemia,leukemia, and the modulation of gastric acid secretion.

The preparation of the compounds cf the invention will now be moreparticularly illustrated by reference to the following experimentalexamples. The purity of compounds was routinely monitored by thin layerchromatography using Merck Kieselgel 60 F₂₅₄ plates. ¹ H-Nuclearmagnetic resonance spectra were recorded using a Nicolet QE-300spectrometer and were in all cases consistent with the proposedstructures.

EXAMPLE 1 2-(N² -Acetyl-N⁶-benzyloxycarbonyl-L-lysyl-aminomethyl)-3-{1-[(cis-4-ethoxycarbonyl-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid t-butyl ester

1-Hydrobenztriazole (207 mg, 1.53 mmole) and N-methylmorpholine (235 mg,2.36 mmole) were added to a stirred solution of N² -acetyl-N⁶-benzyloxycarbonyl-L-lysine (456 mg, 1.41 mmole) in dry dichloromethaneat 0° C., followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (361mg). The solution was stirred at 0° C. for 20 minutes and3-{1-[(cis-4-ethoxycarbonylcyclohexyl)-carbamoyl]cyclopentyl}-2-(aminomethyl)propanoicacid t-butyl ester (500 mg, 1.18 mmole) in dichloromethane (10 ml) wasadded in one portion and the reaction mixture allowed to warm to roomtemperature and stirred for 16 hours. The solution was concentratedunder vacuum to a volume of 10 ml and partitioned between ethyl acetateand water. The organic phase was washed with water (2×50 ml), 2Mhydrochloric acid (50 ml, 2×25 ml), sodium bicarbonate solution (2×25ml) and brine and then dried (MgSO₄) and the solvent evaporated. Theresidue was chromatographed on silica eluting with ethyl acetate to givethe title compound (610 g, 71%). Found: C, 63.34; H, 8.61; N, 7.45. C₃₉H₆₀ N₄ O₉ (0.25 H₂ O) requires C, 63.48; H, 8.33; N, 7.59%.

EXAMPLES 2-27

The following compounds were prepared by the general method of Example 1using as starting materials either3-{1-[cis-4-ethoxycarbonyl-cyclohexyl)carbamoyl]cyclopentyl}-2-(aminomethyl)propanoicacid t-butyl ester (see Preparation 1) or the 3-ethoxycarbonyl isomer(see Preparation 2) and reacting with the appropriate acid of formula IVinstead of N² -acetyl-N⁶ -benzyloxycarbonyl-1-lysine.

    __________________________________________________________________________     ##STR11##                                                                                                     Analysis %                                                             CO.sub.2 C.sub.2 H.sub.5                                                             (Theoretical in brackets)                    Example                                                                            R.sup.6              attachment                                                                           C   H   N                                    __________________________________________________________________________     2                                                                                  ##STR12##           4      66.59 (66.81                                                                      7.81 7.90                                                                         7.23 7.08)                            3                                                                                  ##STR13##           4      66.00 (66.53                                                                      8.38 8.38                                                                         6.86 6.85)                            4                                                                                  ##STR14##           4      69.06 (69.31                                                                      8.16 7.90                                                                         6.18 6.22)                            5                                                                                  ##STR15##           4      61.57 (62.54                                                                      8.64 8.81                                                                         7.69 7.82)                            6                                                                                  ##STR16##           4      64.57 (66.08                                                                      8.21 8.24                                                                         6.68 7.01)                            7                                                                                  ##STR17##           4      63.57 (63.69                                                                      9.11 9.09                                                                         7.26 7.43)                            8                                                                                  ##STR18##           4      66.15 (66.96                                                                      8.67 8.51                                                                         6.85 6.69)                            9                                                                                  ##STR19##           3      64.69 (64.44                                                                      8.94 8.90                                                                         7.27 7.27)                           10                                                                                  ##STR20##           3      67.71 (67.58                                                                      8.24 8.35                                                                         6.34 6.57)                           11                                                                                  ##STR21##           3      62.59 (63.13                                                                      8.80 8.95                                                                         7.63 7.62)                           12                                                                                  ##STR22##           3      66.08 (66.53                                                                      8.49 8.38                                                                         6.75 6.85)                           13                                                                                  ##STR23##           3      66.41 (66.53                                                                      8.35 8.38                                                                         6.63 6.85)                           14                                                                                  ##STR24##           3      63.26 (63.69                                                                      9.28 9.09                                                                         7.16 7.43)                           15                                                                                  ##STR25##           3      61.98 (62.23                                                                      7.78 7.68                                                                         6.27 6.40).sup.(1)                   16                                                                                  ##STR26##           3      64.67 (64.84                                                                      8.28 8.16                                                                         6.59 6.67)                           17                                                                                  ##STR27##           3      67.94 (68.39                                                                      7.81 7.91                                                                         6.38 6.47)                           18                                                                                  ##STR28##           3      67.02 (67.21                                                                      7.57 7.66                                                                         7.20 7.40).sup.(2)                   19                                                                                  ##STR29##           3      66.02 (65.80                                                                      8.08 7.94                                                                         7.94 7.99)                           20                                                                                  ##STR30##           3      65.70 (65.57                                                                      7.98 8.09                                                                         7.46 7.50).sup.(3)                   21                                                                                  ##STR31##           3      64.47 (65.60                                                                      8.21 8.39                                                                         7.22 7.29)                           22                                                                                  ##STR32##           3      68.27 (68.55                                                                      7.77 7.67                                                                         6.56 6.66)                           23                                                                                  ##STR33##           3      63.84 (64.00                                                                      8.13 8.20                                                                         7.37 7.45).sup.(2)                   24                                                                                  ##STR34##           3      58.65 (59.66                                                                      7.84 7.91                                                                         7.17 7.32)                           25                                                                                  ##STR35##           3      63.29 (63.29                                                                      7.73 7.59                                                                         6.49 6.49).sup.(4)                   26                                                                                  ##STR36##           3      63.65 (64.26                                                                      8.39 8.30                                                                         7.63 7.69)                           27                                                                                  ##STR37##           3      66.95 (66.81                                                                      7.95 7.90                                                                         6.75 7.08)                           __________________________________________________________________________     # Z = C.sub.6 H.sub.5 CH.sub.2 OCO-                                           .sup.(1) 0.25 mole CH.sub.3 CO.sub. C.sub.2 H.sub.5                           .sup.(2) 0.5 H.sub.2 O                                                        .sup.(3) 1.0 H.sub.2 O                                                        .sup.(4) 0.5 mole CH.sub.2 Cl.sub.2                                      

EXAMPLES 28-29

The following compounds were prepared following the general method ofExample 1 using as starting material3-{1-[(cis-3-ethoxycarbonylcyclohexyl)carbamoyl]cyclopentyl}-2-amino-propanoicacid and reacting with the appropriate acid of formula IV.

    __________________________________________________________________________     ##STR38##                                                                                              Analysis %                                                                    (Theoretical in brackets)                           Example                                                                            R.sup.6              C   H   N                                           __________________________________________________________________________    28                                                                                  ##STR39##           64.17 (64.10                                                                      7.71 7.69                                                                         8.79 8.90).sup.(1)                          29                                                                                  ##STR40##           67.73 (68.00                                                                      7.62 7.53                                                                         6.16 6.34)                                  __________________________________________________________________________     .sup.(1) hemihydrate                                                     

EXAMPLES 30-33

The following compounds were prepared following the general method ofExample 1 using the appropriate amine of formula (III) and coupling withN² -acetyl-N⁶ -benzyloxycarbonyl-L-lysine.

    ______________________________________                                         ##STR41##                                                                     Example                                                                              ##STR42##         Analysis % (Theoretical in brackets) CHN            ______________________________________                                        30                                                                                    ##STR43##        64.82 (64.60                                                                          8.83 8.66                                                                           7.18 7.18).sup.(1)                     31                                                                                    ##STR44##        63.69 (63.59                                                                          8.43 8.81                                                                           6.94 6.90).sup.(2)                     32                                                                                    ##STR45##        65.95 (65.79 64.52 (64.31                                                             8.90 6.97).sup.(3)                                                                  6.83 7.14)                             33                                                                                    ##STR46##        65.94 (65.79                                                                          8.83 8.73                                                                           7.14 7.14)                             ______________________________________                                         .sup.(1) Hemihydrate                                                          .sup.(2) 1.5 hydrate                                                          .sup.(3) hydrate                                                         

EXAMPLE 34N-[4-{1-[cis-3-t-butoxycarbonylcyclohexyl)-carbamoyl]cyclopentyl}-3-(t-butoxycarbonyl)-butanoyl]phenylalaninet-butyl ester.

1-Hydroxybenztriazole (76 mg, 0.576 mmol) and N-methylmorpholine (0.5ml, 4.5 mmole) were added to a stirred solution of3-{1-[(cis-3-t-butoxycarbonylcyclohexyl)-carbamoyl]cyclopentyl}-2-(carboxymethyl)propanoicacid t-butyl ester (219.6 mg, 0.457 mmol) in dry dichloromethane (20 ml)at 0° C. followed by 1-ethyl-3-(dimethylaminopropyl)carbodimide (94 mg).The solution was stirred at 0° C. for 20 minutes and phenylalaninet-butyl ester (128 mg. 0.5 mmol) in dry dichloromethane (5 ml) added inone portion, and the reaction allowed to warm up to room temperature andstirred for 16 hours. The solution was concentrated under vacuum to avolume of 10 ml, and partitioned between ethyl acetate and water. Theorganic phase was washed with water (2×25 ml), 2M hydrochloric acid(2×10 ml), sodium bicarbonate solution (2×10 ml) and brine, and dried(MgSO₄) and the solvent evaporated to yield the title compound as an oil(325 mg, 100%).

Found: C,66.59; H,8.92; N,4.25. C₃₉ H₆₀ N₂ O₈. H₂ O requires C,66.63;H,8.89; N,3.49%.

EXAMPLES 35-38

The following compounds were prepared following the procedure of Example34 using as starting material the appropriate3-{1-[cis-3-alkoxycarbonyl-cyclohexyl)carbamoyl]cyclopentyl}-2-carboxy-methyl-propanoicacid t-butyl ester t-butyl ester and reacting with the appropriate amineof formula R⁷ R⁹ NH.

    __________________________________________________________________________     ##STR47##                                                                                                Analysis %                                                                    (Theoretical in brackets)                         Example                                                                            R.sup.7          R.sup.16                                                                             C   H   N                                        __________________________________________________________________________    35                                                                                  ##STR48##       C(CH.sub.3).sub.3                                                                   .sup. 63.39 (63.39                                                                8.38 8.38                                                                         6.95 6.98).sup.(1)                        36                                                                                  ##STR49##       C.sub.2 H.sub.5                                                                     .sup. 63.83 (63.96                                                                8.40 8.59                                                                         7.01 6.63)                                37                                                                                  ##STR50##       C.sub.2 H.sub.5                                                                     .sup. 65.17 (65.60                                                                8.39 8.39                                                                         7.10 7.29)                                38                                                                                  ##STR51##       C.sub.2 H.sub.5                                                                     .sup. 63.17 (63.11                                                                7.73 7.54                                                                         8.28 8.48)                                __________________________________________________________________________

EXAMPLE 39 2-(N² -Acetyl-N⁶-benzyloxycarbonyl-L-lysyl-aminomethyl)-3-{1-[(cis-4-ethoxycarbonylcyclohexyl)carbamoyl]cyclopentyl}propanoicacid

The t-butyl ester from Example 1 (571 mg, 0.783 mmol) was dissolved in amixture of trifluoroacetic acid (2 ml) and dichloromethane (1 ml). Thesolution was kept at 4° C. overnight, then concentrated to dryness undervacuum, and the residue was azeotroped six times with dichloromethane.The resulting crude product was then taken up in ethyl acetate andwashed with water until the washings were neutral. The organic phase wasdried (MgSO₄) and evaporated under vacuum to afford the title compound(442 mg, 84%) as a white foam. Found: C,61.89; H,7.76; N,7.93. C₃₅ H₅₂N₄ O₉ (0.5 H₂ O) requires C,61.65; H,7.69; N,8.22%.

EXAMPLES 40 to 65

The following compounds were prepared following the procedure of Example39 but using as starting material the appropriate t-butyl ester ofExamples 2 to 27.

    __________________________________________________________________________     ##STR52##                                                                                                    Analysis %                                                             CO.sub.2 C.sub.2 H.sub.5                                                             (Theoretical in brackets)                     Example                                                                            R.sup.6             attachment                                                                            C   H   N                                    __________________________________________________________________________    40                                                                                  ##STR53##          4      .sup. 65.02 (65.37                                                                7.33 7.41                                                                         7.60 7.61)                            41                                                                                  ##STR54##          4      .sup. 64.18 (64.61                                                                7.54 7.77                                                                         7.63 7.54)                            42                                                                                  ##STR55##          4      .sup. 66.94 (67.83                                                                7.34 7.32                                                                         6.65 6.78)                            43                                                                                  ##STR56##          4      .sup. 49.96 (49.69                                                                6.49 6.26                                                                         6.50 6.44).sup.(1)                    44                                                                                  ##STR57##          4      .sup. 63.67 (64.07                                                                7.80 7.60                                                                         7.52 7.73)                            45                                                                                  ##STR58##          4      .sup. 60.89 (61.27                                                                8.58 8.50                                                                         8.14 8.25)                            46                                                                                  ##STR59##          4      .sup. 64.88 (65.12                                                                7.95 7.93                                                                         7.34 7.35)                            47                                                                                  ##STR60##          3      .sup. 62.11 (62.16                                                                8.44 8.31                                                                         7.95 8.06)                            48                                                                                  ##STR61##          3      .sup. 65.49 (65.85                                                                7.80 7.77                                                                         6.99 7.20)                            49                                                                                  ##STR62##          3      .sup. 60.20 (60.58                                                                8.46  8.34                                                                        8.30 8.48)                            50                                                                                  ##STR63##          3      .sup. 64.86 (64.61                                                                7.91 7.77                                                                         7.24 7.54)                            51                                                                                  ##STR64##          3      .sup. 64.61 (64.61                                                                7.96 7.77                                                                         7.33 7.54)                            52                                                                                  ##STR65##          3      .sup. 61.08 (61.27                                                                8.52 8.50                                                                         7.91 8.25)                            53                                                                                  ##STR66##          3      .sup. 60.24 (60.25                                                                7.19 6.97                                                                         6.90 7.27)                            54                                                                                  ##STR67##          3      .sup. 62.19 (62.32                                                                7.59 7.58                                                                         7.15 7.27).sup.(2)                    55                                                                                  ##STR68##          3      .sup. 66.10 (66.09                                                                7.21 7.33                                                                         6.97 7.01).sup.(3)                    56                                                                                  ##STR69##          3      .sup. 63.70 (63.64                                                                6.87 6.98                                                                         7.53 7.56).sup.(4)                    57                                                                                  ##STR70##          3      .sup. 63.83 (63.75                                                                7.59 7.54                                                                         8.32 8.45).sup.(5)                    58                                                                                  ##STR71##          3      .sup. 65.41 (65.63                                                                7.66 7.62                                                                         8.01 8.14)                            59                                                                                  ##STR72##          3      .sup. 62.16 (64.02                                                                7.95 7.92                                                                         7.62 7.80)                            60                                                                                  ##STR73##          .sup. 3.sup.(8)                                                                      gum Rf 0.33 (silica)                          61                                                                                  ##STR74##          3      .sup. 58.34 (58.25                                                                7.38 7.41                                                                         8.60 8.77).sup.(6)                    62                                                                                  ##STR75##          3      .sup. 57.40 (57.61                                                                7.48 7.40                                                                         7.89 7.90)                            63                                                                                  ##STR76##          3      .sup. 63.20 (63.02                                                                7.47 7.25                                                                         7.23 7.13)                            64                                                                                  ##STR77##          3      .sup. 60.54 (60.79                                                                7.81 7.63                                                                         7.94 8.07)                            65                                                                                  ##STR78##          3      .sup. 64.53 (64.79                                                                 7.29 7.35                                                                        7.36 7.54).sup.(9)                    __________________________________________________________________________     .sup.(1) 1.5 mole CF.sub.3 CO.sub.2 H                                         .sup.(2) 0.25 mole H.sub.2 O                                                  .sup.(3) 0.33 mole H.sub.2 O                                                  .sup.(4) 0.5 mole CH.sub.2 Cl.sub.2                                           .sup.(5) 0.5 mole H.sub.2 O                                                   .sup.(6) HCl in CH.sub.2 Cl.sub.2 at 0° C. for 3 hours used instea     of trifluoroacetic acid. Product isolated as .HCl.H.sub.2 O.                  .sup.(7) 0.1 mole CH.sub.2 Cl.sub.2.                                          .sup.(8) From Example 106.                                               

EXAMPLES 66-67

The following compounds were prepared following the general method ofExample 39 using as starting material the appropriate t-butyl ester ofExamples 28 and 29.

    __________________________________________________________________________     ##STR79##                                                                                              Analysis %                                                                    (Theoretical in brackets)                           Example                                                                            R.sup.6              C   H   N                                           __________________________________________________________________________    66                                                                                  ##STR80##           .sup. 61.79 (62.07                                                                7.19 7.20                                                                         9.39 9.52).sup.(1)                          67                                                                                  ##STR81##           foam                                                __________________________________________________________________________     .sup.(1) 0.75 H.sub.2 O                                                  

EXAMPLES 68-71

The following compounds were prepared by the general method of Example39 using the appropriate t-butyl ester of Examples 30 to 33.

    ______________________________________                                         ##STR82##                                                                     Example                                                                              ##STR83##         CHN(Theoretical in brackets)Analysis                ______________________________________                                                                 %                                                    68                                                                                    ##STR84##        .sup. 60.74 (61.05                                                                    7.97 7.85                                                                           7.42 7.40).sup.(1)                     69                                                                                    ##STR85##        .sup. 62.64 (62.71                                                                     8.22 8.37                                                                          7.47 7.50)                             70                                                                                    ##STR86##        two diastereoisomers isolated as gums                71                                                                                    ##STR87##        gum                                                  ______________________________________                                    

EXAMPLES 72-76

The following compounds were prepared by the general method of Example39 using the appropriate t-butyl ester of Examples 34-38.

    ______________________________________                                         ##STR88##                                                                                             Analysis %                                           Ex-                      (Theoretical in brackets)                            ample R.sup.7         R.sup.4                                                                              C     H     N                                    ______________________________________                                        72                                                                                   ##STR89##      H      .sup. 61.49 (62.13                                                                  7.29 7.19                                                                           4.64 4.28).sup.(1)                   73                                                                                   ##STR90##      H      .sup. 60.98 (60.95                                                                  7.47 7.71                                                                           7.86 8.36).sup.(2)                   74                                                                                   ##STR91##      C.sub.2 H.sub.5                                                                      .sup. 59.78 (59.79                                                                  7.65 7.53                                                                           7.14 7.40)                           75                                                                                   ##STR92##      C.sub.2 H.sub.5                                                                      .sup. 60.58 (60.70                                                                  7.55 7.55                                                                           7.23 7.33)                           76                                                                                   ##STR93##      C.sub.2 H.sub.5                                                                      .sup. 62.01 (61.81                                                                  7.01 7.08                                                                           8.86 9.16)                           ______________________________________                                         .sup.(1) 0.5 mole CH.sub.2 CO.sub.2 C.sub.2 H.sub.5                           .sup.(2) 0.6 H.sub.2 O                                                   

EXAMPLE 77 2-(N² -Acetyl-N⁶-benzyloxycarbonyl-L-lysyl-aminomethyl)-3-{1-[(cis-4-carboxycyclohexyl)carbamoyl]cyclopentyl}propanoicacid

The ethyl ester from Example 39 (404 mg, 0.600 mmol) was dissolved in 2Msodium hydroxide solution (10 ml), and the resulting solution was keptat room temperature overnight. The reaction mixture was diluted to 30 mland extracted with diethyl ether. The aqueous phase was acidified to pH1 with 2M hydrochloric acid and extracted with ethyl acetate (3×25 ml).The organic phase was dried (MgSO₄) and evaporated to afford the titlecompound (375 mg, 97%) as a white foam. Found: C,58.30; H,7.24; N,8.20.C₃₃ H₄₈ N₄ O₉. (0.25 CH₂ Cl₂, 1.25 H₂ O) requires C,58.00; H,7.17;N,8.14%.

EXAMPLES 78-96

The following compounds were prepared following the procedure of Example77 but using as starting material the appropriate ethyl ester ofExamples 40 to 58.

    __________________________________________________________________________     ##STR94##                                                                                                    Analysis %                                                              CO.sub.2 H                                                                          (Theoretical in brackets)                     Example                                                                            R.sup.6              attachment                                                                          C   H  N                                      __________________________________________________________________________    78                                                                                  ##STR95##           4     63.42 (63.94                                                                      7.16 7.34                                                                        7.48 7.68.sup.(1)                      79                                                                                  ##STR96##           4     61.34 (61.50                                                                      7.47 7.39                                                                        7.57 7.65).sup.(2)                     80                                                                                  ##STR97##           4     65.39 (65.49                                                                      6.84 7.08                                                                        6.92 6.94).sup.(3)                     81                                                                                  ##STR98##           4     52.87 (52.67                                                                      6.66 6.97                                                                        8.34 8.01).sup. (4)                    82                                                                                  ##STR99##           4     60.78 (60.82                                                                      7.19 7.11                                                                        7.73 7.82).sup.(5)                     83                                                                                  ##STR100##          4     58.16  (58.16                                                                     8.03 8.08                                                                        8.18 8.43).sup.(6)                     84                                                                                  ##STR101##          4     63.30 (63.49                                                                      7.75 7.70                                                                        7.17 7.25).sup.(7)                     85                                                                                  ##STR102##          3     59.52 (59.74                                                                      8.04 8.02                                                                        8.07 8.36).sup.(1)                     86                                                                                  ##STR103##          3     64.68 (64.84                                                                      7.62 7.44                                                                        7.15 7.56)                             87                                                                                  ##STR104##          3     55.66 (56.09                                                                      7.71 7.80                                                                        8.31 8.44).sup.(8)                     88                                                                                  ##STR105##          3     62.77 (63.13                                                                      7.57 7.63                                                                        7.31 7.44).sup.(9)                     89                                                                                  ##STR106##          3     62.38 (62.43                                                                      7.47 7.49                                                                        7.60 7.80).sup.(1)                     90                                                                                  ##STR107##          3     58.73 (58.75                                                                      8.21 8.22                                                                        8.32 8.57).sup.(1)                     91                                                                                  ##STR108##          3     57.76 (58.00                                                                      6.66 6.67                                                                        7.30 7.52).sup.(1)                     92                                                                                  ##STR109##          3     60.81 (60.63                                                                      7.25 7.27                                                                        7.57 7.58).sup.(1)                     93                                                                                  ##STR110##          3     64.52 (64.48                                                                      7.15 7.08                                                                        6.98 7.09).sup.(10)                    94                                                                                  ##STR111##          3     65.12 (65.41                                                                      7.07 7.01                                                                        7.88  8.12).sup.(1)                    95                                                                                  ##STR112##          3     63.19 (62.98                                                                      7.27 7.30                                                                        8.11 8.74).sup.(1)                     96                                                                                  ##STR113##          3     64.24 (64.26                                                                      7.42 7.43                                                                        8.11 8.33).sup.(1)                     __________________________________________________________________________     .sup.(1) 0.5 mole H.sub.2 O;                                                  .sup.(2) 0.125 mole CH.sub.2 Cl.sub.2 ; 0.5 mole H.sub.2 O;                   .sup.(3) 0.75 mole H.sub.2 O;                                                 .sup.(4) 0.5 mole CH.sub.2 Cl.sub.2 ; 0.25 mole CF.sub.3 CO.sub.2 H;          .sup.(5) 0.2 mole CH.sub.2 Cl.sub.2 ; 0.25 mole H.sub.2 O;                    .sup.(6) 0.125 mole CH.sub.2 Cl.sub.2 ; 0.33 mole H.sub.2 O;                  .sup.(7) 0.4 mole CH.sub.3 CO.sub.2 C.sub.2 H.sub.5 ;                         .sup.(8) 0.25 mole CH.sub.2 Cl.sub.2 ; 0.5 mole H.sub.2 O;                    .sup.(9) 0.28 mole CH.sub.3 CO.sub.2 C.sub.2 H.sub.5 ; 0.14 mole H.sub.2      O;                                                                            .sup.(10) 0.2 mole CH.sub.3 CO.sub.2 C.sub.2 H.sub.5 ; 0.5 mole H.sub.2 O                                                                              

EXAMPLE 97 2-(N²-Acetyl-L-lysyl-aminomethyl)-3-{1-[(cis-4-carboxycyclohexyl)carbamoyl]cyclopentyl}propanoicacid

A solution of the product from Example 77 (200 mg, 0.31 mmol) in amixture of ethanol (27 ml) and water (3 ml) was reduced on 10% palladiumon charcoal (20 mg) under 50 p.s.i. (3.46 bar) of hydrogen for 11/2hours. The solution was filtered and the solvent evaporated undervacuum, and the residue azeotroped with dichloromethane (6×) to affordthe title compound (161 mg, 100%) as a white solid, m.p. 161°-163° C.Found: C,56.80; H,8.49; N,9.22. C₂₅ H₄₂ N₄ O₇.H₂ O requires C,56.80;H,8.39; N,10.60%.

EXAMPLES 98-100

The following compounds were prepared following the procedure of Example97 but using as starting material the appropriate amino-protected ethylester or acid.

    __________________________________________________________________________     ##STR114##                                                                                                   Analysis %                                                              CO.sub.2 R.sup.4                                                                    (Theoretical in brackets)                     Example                                                                            R.sup.6              attachment                                                                          C   H  N                                      __________________________________________________________________________     98                                                                                 ##STR115##          4-CO.sub.2 H                                                                        60.64 (60.58                                                                      7.84 7.79                                                                        8.77 9.42.sup.(1)                       99                                                                                 ##STR116##          3-CO.sub.2 C.sub.2 H.sub.5                                                          64.48 (64.29                                                                      7.96 7.76                                                                        8.21 9.14).sup.(1)                     100                                                                                 ##STR117##          3-CO.sub.2 H                                                                        63.57 (63.48                                                                      7.68 7.51                                                                        8.46 9.49).sup.(1)                     101                                                                                 ##STR118##          3-CO.sub.2 C.sub.2 H.sub.5                                                          61.74 (61.43                                                                      8.27 8.16                                                                        9.20 9.95.sup.(1)                      102                                                                                 ##STR119##          3-CO.sub.2 H                                                                        60.54 (60.42                                                                      7.93 7.91                                                                        9.97 10.36.sup.(1)                     103                                                                                 ##STR120##          3-CO.sub.2 C.sub.2 H.sub.5                                                          64.03 (64.13                                                                      8.41 8.21                                                                        8.48 9.59).sup.(2)                     104                                                                                 ##STR121##          3-CO.sub.2 H                                                                        62.16 (62.08                                                                      8.08 8.03                                                                        9.58 9.79).sup.(1)                     105                                                                                 ##STR122##          3-CO.sub.2 C.sub.2 H.sub.5                                                          58.87 (60.88                                                                      8.50 8.53                                                                        8.88 9.41).sup.(3)                     106                                                                                 ##STR123##          3-CO.sub.2 C.sub.2 H.sub.5                                                          gum                                           107                                                                                 ##STR124##          3-CO.sub.2 C.sub.2 H.sub.5                                                          51.75 (52.50                                                                      8.07 7.89                                                                        8.83 9.33).sup.(4)                     108                                                                                 ##STR125##          3-CO.sub.2 C.sub.2 H.sub.5                                                          57.07 (59.15                                                                      8.85 8.84                                                                        10.12 10.99).sup.(5)                   109                                                                                 ##STR126##          3-CO.sub.2 C.sub.2 H.sub.5                                                          55.63 (57.32                                                                      8.42 8.73                                                                        9.27 9.91).sup.(6)                     110                                                                                 ##STR127##          3-CO.sub.2 C.sub.2 H.sub.5                                                          62.46 (62.12                                                                      8.17 8.15                                                                        8.65 9.06).sup.(1)                     __________________________________________________________________________     .sup.(1) Hydrate                                                              .sup.(2) 0.25H.sub.2 O                                                        .sup.(3) 0.2 mole CH.sub.2 Cl.sub.2                                           .sup.(4) 0.25 mole H.sub.2 O, 0.25 mole CH.sub.2 Cl.sub.2                     .sup.(5) 0.25 mole H.sub.2 O, 0.1 CH.sub.2 Cl.sub.2                           .sup.(6) From Example 22, tbutyl ester.                                  

EXAMPLES 111-112

The following compounds were prepared from Examples 66 and 67 by thehydrolysis of the ester group following the general procedure of Example77 followed by catalytic hydrogenation or by treatment with HBr inglacial acetic acid to remove the benzyloxycarbonyl protecting group.

    ______________________________________                                         ##STR128##                                                                                             Analysis %                                          Ex-                       (Theoretical                                        am-                       in brackets)                                        ple  R.sup.6              C       H    N                                      ______________________________________                                        111                                                                                 ##STR129##          .sup. 59.24 (59.14                                                                    7.62 7.44                                                                          12.15 12.32).sup.(1)                   112                                                                                 ##STR130##          foam                                                ______________________________________                                         .sup.(1) 0.5 mole H.sub.2 O                                              

EXAMPLES 113-119

The following Examples were prepared from Examples 73 to 76 by catalytichydrogenation according to the procedure of Example 97, followed, in thecase of the ethyl esters, by hydrolysis according to the procedure ofExample 77. The diacids were isolated by ion-exchange chromatographyeluting with aqueous pyridine.

    ______________________________________                                         ##STR131##                                                                                            Analysis %                                           Ex-                      (Theoretical in brackets)                            ample R.sup.7         R.sup.4                                                                              C     H     N                                    ______________________________________                                        113                                                                                  ##STR132##     H      .sup. 55.62 (55.62                                                                  8.37 9.57                                                                           7.95 7.95).sup.(1)                   114 115                                                                              ##STR133##     C.sub.2 H.sub.5 H                                                                    .sup. 60.10 (59.82 .sup. 56.27 (56.42                                               8.71 8.79 8.37 8.77                                                                 8.34 8.67  9.79 9.74)                116 117                                                                              ##STR134##     C.sub.2 H.sub. 5 H                                                                   .sup. 59.52 (59.35 59.14 (58.97                                                     8.51 8.76 8.46 8.53                                                                 8.53 8.92) 9.35 9.48                 118 119                                                                              ##STR135##     C.sub.2 H.sub.5 H                                                                    .sup. 59.02 (58.88 56.79 (60.72                                                     7.81 7.75 8.01 7.55                                                                 10.55 10.33) 9.08 12.21)             ______________________________________                                         .sup.(1) 3.3 moles C.sub.2 H.sub.5 OH, 1.5 mole H.sub.2 O                

EXAMPLES 120-125

The following compounds were prepared by hydrolysis of the appropriateester of Examples 61 or 105-110 following the procedure of Example 77.The products were isolated by ion-exchange chromatography eluting withaqueous pyridine.

    __________________________________________________________________________     ##STR136##                                                                                            Analysis %                                                                    (Theoretical in brackets)                            Example                                                                            R.sup.6             C   H  N                                             __________________________________________________________________________    120                                                                                 ##STR137##         57.36 (59.13                                                                      8.29 8.51                                                                        9.28 9.85).sup.(1)                            121                                                                                 ##STR138##         64.43 (64.64                                                                      7.60 7.50                                                                        8.95 8.87).sup.(2)                            122                                                                                 ##STR139##         61.54 (61.58                                                                      7.59 7.30                                                                        9.90 9.90).sup.(2)                            123                                                                                 ##STR140##         50.32 (52.38                                                                      7.86 7.77                                                                         9.04 10.18).sup.(3)                          124                                                                                 ##STR141##         53.61 (57.30                                                                      8.55 8.68                                                                        10.69 11.62).sup.(4)                          125                                                                                 ##STR142##         56.57 (56.79                                                                      8.55 8.39                                                                        10.19 10.60).sup.(1)                          126                                                                                 ##STR143##         60.85 (60.67)                                                                     7.96 8.05                                                                        8.69 9.13).sup.(5)                            __________________________________________________________________________    __________________________________________________________________________     .sup.(1) hydrate                                                              .sup.(2) 0.5 mole H.sub.2 O                                                   .sup.(3) 0.2 mole H.sub.2 O                                                   .sup.(4) 0.75 mole H.sub.2 O                                                  .sup.(5) 1.0 mole H.sub.2 O 0.5 mole C.sub.2 H.sub.5 OH                  

EXAMPLES 127-134

The following compounds were prepared from Examples 68-71 by catalytichydrogenation according to the procedure of Example 97 to yield theesters (R₄ =CH₃ or C₂ H₅) followed by hydrolysis following the procedureof Example 77 to yield the corresponding acids (R⁴ =H).

    __________________________________________________________________________     ##STR144##                                                                    Example                                                                             ##STR145##   R.sup.4                                                                          ##STR146##                                             __________________________________________________________________________     127 128                                                                             ##STR147##   CH.sub.3 H                                                                         59.19 (58.85 60.12 (60.01                                                         8.93 9.13 9.00 8.94                                                               8.62 9.15).sup.(1) 9.88 9.65).sup.(2)          129 130                                                                            ##STR148##   C.sub.2 H.sub.5 H                                                                  60.05 (60.13 58.59 (58.54                                                         8.97 8.81 8.97 9.08                                                               8.55 8.98).sup.(3) 8.85 9.26).sup.(4)         .sup.(1) 1.75 H.sub.2 O                                                       .sup.(2) 0.75 H.sub.2 O                                                       .sup.(3) 0.4 mole CH.sub.2 Cl.sub.2                                           .sup.(4) 1.5 H.sub.2 O, 0.25 C.sub.2 H.sub.5 OH                               131 132                                                                             ##STR149##   C.sub.2 H.sub.5 H                                                                ##STR150##                                              133 134                                                                             ##STR151##   C.sub.2 H.sub.5 H                                                                ##STR152##                                             __________________________________________________________________________     .sup.(1) 0.5 H.sub.2 O                                                   

EXAMPLE 135 2-(N²-Methanesulphonyl-L-lysyl-aminomethyl)-3-{1-[(cis-4-carboxy-cis-3-butylcyclohexyl)carbamoyl-cyclopentyl}propanoicacid

1. 3-(1-Carboxycyclopentyl)-2-aminopropanoic acid t-butyl ester

3-(1-Carboxycyclopentyl)-2-(dibenzylaminomethyl)propanoic acid t-butylester hydrochloride (20.0 g; 41 mmol) in ethanol (160 ml) andtriethylamine (2.0 ml) was hydrogenated over palladium (from 20% Pd(OH)₂/C; 20 g) at 60 p.s.i. (4.1 bar). After eighteen hours the mixture wasfiltered through arbicel, the solvent evaporated and the residue driedazeotropically with toluene. The required primary amino acidtriethylamine salt, containing one mole equivalent of triethylaminehydrochloride was thus obtained as a white solid (16.21 g).

2. 2-(N² -Benzyloxycarbonyl-N⁶-t-butoxycarbonyl-L-lysylaminomethyl)-3-[1-(1-carboxycyclopentyl)]propanoicacid t-butyl ester (diastereoisomer)

The above product (8.24 g; 20.1 mmole) and N² -benzyloxycarbonyl-N⁶-t-butoxycarbonyl-L-lysine 4-nitrophenyl ester (9.50 g, 18.9 mmole) weredissolved in dry methylene chloride (70 ml). The solution was stirredand after cooling to 10° C., triethylamine (2.64 ml, 18.9 mmol) wasadded. After half an hour the mixture became homogenous and was allowedto stand at room temperature overnight. The solution was then washedwith 1M citric acid followed by water, dried over MgSO₄ and evaporated.The residue was purified by chromatography on silica gel (500 g) elutingwith increasing proportions of ethyl acetate in hexane (2:1 to 4:1) andfinally with ethylacetate, hexane, acetic acid (4:1:0.05). The requiredmixture of diastereoisomers was thus obtained as a colourless gum (10.5g). The isomers were then separated by chromatography on silica gel (1kg) eluting with a mixture of toluene, isopropanol and diethylamine(10:2:1). The diethylamine salt of the required more polardiastereoisomer was obtained as an orange foam (3.01 g), which wasdissolved in ethyl acetate and washed with 1M citric acid and brine.Drying over MgSO₄ and evaporation gave the free acid as a yellow foam(2.81 g). Found: C,62.89; H,8.29; N,6.69. C requires C,62.54; H,8.11;N,6.63%. Additional chromatography of a small sample on silica elutingwith increasing proportions of ethyl acetate in hexane (2:3 to 17:3)gave a cream powder [α]_(D) ²⁵ -2.8°, [α]₃₆₅ ²⁵ -3.6° (c=0.5, ethanol).

3. 2-(N² -Benzyloxycarbonyl-N⁶-t-butoxycarbonyl-L-lysylaminomethyl)-3-{1-[(cis-4-ethoxycarbonyl-cis-3-butylcyclohexyl)carbamoyl]-cyclopentyl}propanoicacid t-butyl ester

Coupling of the above product from step 2 (650 mg; 1.03 mmole) withc-4-amino-c-2-butyl-r-1-cyclohexane carboxylic acid ethyl esterhydrochloride (271 mg; 1.03 mmole) as described in Example 1 followed bychromatography on silica eluting with increasing proportions of ethylacetate in hexane (7:3 to 4:6) gave the required product as a pale foam(630 mg; 73%). Found: C,64 37; H,8.98; N,6.81. C₄₆ H₇₄ N₄ O₁₀ (0.75 H₂O) requires C,64.50; H,8.88; N,6.54%.

4. 2-(N² -Methanesulphonyl-L-lysyl-aminomethyl)-3-}1-[(cis-4-carboxycis-3-butylcyclohexyl)carbamoyl]cyclopentyl}propanoic acid

(i) The above product from step 3 (620 mg; 0.735 mmole) in ethanol (18ml) and water (2 ml) was hydrogenated over 5% palladium on carbon (200mg) at 50 p.s.i. (3.5 bar). After three hours the mixture was filteredthrough Arbicel and evaporated to dryness giving a white foam (520 mg;95%).

(ii) Methane sulphonyl chloride (0.11 ml; 1.41 mmole) was added dropwiseto an ice cold stirred solution of the above product (500 mg; 0.67mmole) and N-methylmorpholine (0.16 ml; 1.4 mmole) in dry methylenechloride (15 ml). After three hours more methane sulphonyl chloride(0.03 ml) and N-methylmorpholine (0.04 ml) were added and the mixturekept at 0° C. overnight. The mixture was then washed in succession withwater, saturated aqueous sodium bicarbonate and water, dried over MgSO₄and evaporated to give the crude product which was chromatographed onsilica gel. Elution with increasing proportions of ethyl acetate inhexane (4:6 to 1:9) gave the required methanesulphonyl derivative as acolourless foam (460 mg; 87%). Rf. 0.15 (ethylacetate, hexane, 1:1).

(iii) Treatment of the above product (440 mg; 0.56 mmole) withtrifluoroacetic acid as described in Example 39, followed by hydrolysiswith 1N sodium hydroxide (4.5 ml) at 50°-55° C. for 65 hours andadsorption on ion-exchange resin 50W-X8 eluting with 10% aqueouspyridine gave the required diacid as a foam. Trituration withacetonitrile afforded a white powder (245 mg; 73%). Found: C,54.92;H,8.29; N,9 12. C₂₈ H₅₀ N₄ O₈ S (0.5 H₂ O) requires C,54.97; H,8.40;N,9.16%.

EXAMPLE 136 2-(N²-Methanesulphonyl-L-lysyl-aminomethyl)-3-{1-[(cis-4-carboxy-cis-3-(3-methylbutyl)-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid

This compound was prepared following the procedure of Example 135 butusing c-4-amino-c-2-(3-methylbutyl)-r-1-cyclohexane carboxylic acid instep 3. The product was obtained as a cream powder. Found: C,56.07;H,8.22; N,8.95. C₂₉ H₅₂ N₄ O₈ S requires C,56.47; H,8.50; N,9.08%.

PREPARATION 13-{1[(cis-4-Ethoxycarbonyl-cyclohexyl)carbamoyl]cyclopentyl}-2-(aminomethyl)propanoicacid t-butyl ester

(a) A solution of 2-(bromomethyl)propenoic acid t-butyl ester (20 g,90.5 mmole) in dry acetonitrile (360 ml), cooled to 0° C., was treatedwith solid potassium carbonate (15.63 g, 113 mmole), followed by asolution of dibenzylamine (17.83 g, 90.5 mmole, in dry acetonitrile (600ml), producing a 10° C. exotherm. The reaction was stirred at 0° C. for0.5 hours, followed by 1 hour at room temperature, and then partitionedbetween water and diethyl ether. The ether phase was washed again withwater, dried (sodium sulphate) and evaporated to yield the crude product(31 g) which was filtered through a pad of silica, eluting withhexane/CH₂ Cl₂ (1:1), to yield 2-(dibenzylaminomethyl)propenoic acidt-butyl ester (23.8 g, 78%) as a solid, m.p. 62°-63° C. Found C,78.09;H,8.20; N,4.18. C₂₂ H₂₇ NO₂ requires C,78.3; N,8.06; N,4.15 %.

(b) To a stirred solution of diisopropylamine (14.98 g, 20.75 ml, 148mmole) in dry tetrahydrofuran (250 ml) cooled to -30° C. under nitrogen,was added dropwise, n-butyl lithium (59.3 ml of a 2.5M solution, 148mmole), keeping the temperature below -20° C. The reaction was stirredat -20° C. for 1 hour, then cooled to -30° C. and cyclopentanecarboxylicacid (8.05 g, 7.65 ml, 70.6 mmole) added dropwise in a small amount ofdry tetrahydrofuran. The reaction mixture was stirred at 0° C. for twohours, during which time a white precipitate formed. The solution wasthen cooled to -70° C., and a solution of2-(dibenzylaminomethyl)-propenoic acid t-butyl ester (23.8 g, 70.6 mmol)in dry tetrahydrofuran (35 ml) was added dropwise. The reaction was leftovernight (below -40° C.), and then poured into iced hydrochloric acid(4.2 eq, final pH=2) and the product extracted into diethyl ether. Theether layer was washed with hydrochloric acid, water (2×), dried (Na₂SO₄) and evaporated to yield the crude product (40 g). This waschromatographed over silica gel (500 g) eluting with dichloromethanecontaining methanol (0 to 5%) to yield, on evaporation of the purefractions, 3-(1-carboxycyclopentyl)-2(dibenzylaminomethyl)propanoic acidt-butyl ester as an oil (18.3 g, 57%). Found: C,74 38; H,8.41; N,2 91.C₂₈ H₃₇ NO₄ requires C,74.47; H,8.26; N,3.10%.

(c) The t-butyl ester from step (b) above (11.97 g, 26.54 mmole) in drydichloromethane (200 ml), under nitrogen, was treated with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (10.19 g, 53mmole), 1-hydroxybenztriazole (3.94 g, 29 mmole), triethylamine (16.08g, 22.15 ml, 159 mmole) and cis-4-aminocyclohexane-carboxylic acid ethylester hydrochloride (6.04 g, 20 mmole). After stirring overnight at roomtemperature, the solvent was evaporated and the residue partitionedbetween ethyl acetate and water. The organic phase was washed withdilute hydrochloric acid (2×), dilute aqueous sodium bicarbonate, water,dried (Na₂ SO₄) and evaporated to yield the crude product (15.5 g). Thiswas chromatographed on silica (400 g) eluting with 1 to 10% diethylether in dichloromethane. Evaporation of the desired fractions gave3-{1-[(cis-4-ethoxycarbonyl-cyclohexyl)-carbamoyl]cyclopentyl}-2-(dibenzylaminomethyl)propanoicacid t-butyl ester as an oil (11.43 g, 71%). Found: C,73.38; H,8.87;N,4.42. C₃₇ H₅₂ N₂ O₅ requires C,73.48; H,8.67; N,4.68%.

(d) The product from step (c) above (4.6 g, 7.62 mmole) in ethanol (75ml) was hydrogenated in an atmosphere of hydrogen (50 p.s.i., 3.46 bar)over palladium on charcoal (200 mg), at room temperature overnight. Thereaction mixture was filtered through a Solkaflok pad, and the filtrateevaporated to dryness to yield the crude product as an oil. This wasdissolved in diethyl ether, and extracted into dilute hydrochloric acid(pH 2) (2×). The combined aqueous layers were washed with diethyl ether,then neutralised with saturated aqueous sodium bicarbonate and extractedwith dichloromethane (3×). The combined organic phases were dried (Na₂SO₄) and evaporated to yield the title amine as an oil (2.91 g, 90%).Found: C,64.80; H,9.28; N,6.76. C₂₃ H₄₀ N₂ O₅ requires C,65.06; H,9.50;N,6.60%.

PREPARATIONS 2-6

The following compounds were prepared following the procedure ofPreparation 1 but using the appropriate amino-cycloalkane carboxylicacid ester in step (c) instead of cis-4-aminocyclohexane-carboxylic acidethyl ester.

    ______________________________________                                         ##STR153##                                                                    Preparation                                                                           ##STR154##       Analysis % (Theoretical in brackets) CHN            ______________________________________                                                 ##STR155##      60.46 (61.88                                                                           9.11 6.09 6.19)                             3                                                                                      ##STR156##      66.34 (66.28                                                                          10.02 5.67 5.95).sup.(1)                     4                                                                                      ##STR157##      gum Rf 0.69 (silica; CH.sub.2 Cl.sub.2, CH.sub.3                              OH, CH.sub.3 CO.sub.2 H, 90:10:1)                    5                                                                                      ##STR158##      67.10 (67.46                                                                          10.09 10.06                                                                         5.69 5.83)                             6                                                                                      ##STR159##      67.07 (67.46                                                                          10.06 10.06                                                                         5.71 5.83)                             ______________________________________                                         .sup.(1) 0.25 mole H.sub.2 O                                             

PREPARATION 7 c-4-Amino-c-2-butyl-r-1-cyclohexane carboxylic acid ethylester hydrochloride

(1) cis-2-Butyl-4-oxocyclohexane carboxylic acid ethyl ester2-Butyl-4-oxocyclohex-2-ene carboxylic acid ethyl ester [Tetrahedron 371033 (1981)] (4.48 g; 20 mmole) dissolved in absolute ethanol (15 ml)containing 2N hydrochloric acid (1 ml) was reduced at room temperatureover 5% palladium on carbon (150 mg) at 50 p.s.i. (3.45 bar). After onehour the mixture was filtered through avicel and the solvent wasevaporated under reduced pressure. The residue was taken up in diethylether and washed successively with water, saturated aqueous sodiumbicarbonate and water. Drying over MgSO₄ and evaporation gave an oil(4.2 g) which was chromatographed on silica. Elution with diethylether:hexane (2:8) gave the pure title ester (3.6 g, 80%) as a clearliquid. Found: C, 68.86; H, 9.84. C₁₃ H₂₂ O₃ requires C, 68.99; H,9.80%.

(2) cis-2-Butyl-4-hydroximinocyclohexane carboxylic acid ethyl ester

Sodium acetate (1.56 g; 19 mmole) and hydroxylamine hydrochloride (1.32g; 19 mmole) were dissolved in water (5 ml). Ethanol (50 ml) was addedand the mixture was filtered. The above ester (3.57 g; 15.8 mmole) wasadded and the solution was refluxed for two hours. The solvent wasevaporated under reduced pressure and the residue partitioned betweendiethyl ether and water. The ether extract was washed in turn withsaturated aqueous sodium bicarbonate and water, dried (MgSO₄) and thesolvent evaporated to give the required oxime as an oil (3.8 g; 100%).Found: C, 64.64; H, 9.48; N, 5.91. C₁₃ H₂₃ NO₃ requires C, 64.70; H,9.61; N, 5.80%.

(3) c-2-Butyl-c-4-(1,1-dimethylethoxycarbonylamino-r-1-cyclohexanecarboxylic acid ethyl ester

An aqueous solution of titanium trichloride (37 ml, 15% w/v; 36.5 mmole)was added dropwise at room temperature under nitrogen over 1.5 hours toa stirred solution of the above oxime (4.0 g; 16.57 mmole), ammoniumacetate (16 g) and sodium cyanoborohydride (3.12 g; 49.7 mmole) inabsolute ethanol (200 ml). After stirring for 14 hours the solvent wasevaporated, water was added and the mixture was basified to pH 8 with 1Nsodium hydroxide. The product was stirred in air to oxidize unreactedreagent, and the suspension was then extracted with ethyl acetate. Theorganic extract was washed with saturated salt solution, dried (MgSO₄)and evaporated to give the crude amine as a clear gum (4.3 g). Thisproduct was dissolved in dry methylene chloride (80 ml) containingN-methylmorpholine (1.68 g; 16.6 mmole), di-tert-butyldicarbonate (7.23g; 33.14 mmole) added and the solution allowed to stand at roomtemperature for 48 hours. The solvent was evaporated and the residuepartitioned between diethyl ether and water. The ether extract waswashed successively with 0.5N hydrochloric acid, water, saturatedaqueous sodium bicarbonate and water. Drying over MgSO₄ and evaporationgave an oil (5.8 g) which was chromatographed on silica (600 g). Elutionwith diethyl ether:hexane (2:8) gave the required cis compound as an oil(2.72 g; 50%). Rf 0.38 (silica; ether:hexane 3:7). NMR: δ=2.66 (HC--CO₂Et), δ=3.45 (HC--NH--). Found C, 66.06; H, 10.17; N, 4.19. C₁₈ H₃₃ NO₄requires C, 66.02; H, 10.16; N, 4.28%. Continued elution then gave themore polar trans-isomer as an oil which solidified on standing (860 mg;16%) Rf 0.27. NMR: δ=2.45 (HC--CO₂ Et), δ=3.68 (HC--NH--). Found C,65.75; H, 10.13; N, 4.17. C₁₈ H₃₃ NO₄ requires C, 66.02; H, 10.16; N,4.28%.

(4) c-4-Amino-c-2-butyl-r-1-cyclohexane carboxylic acid ethyl esterhydrochloride

An ice cold solution of the above cis-isomer (3.2 g; 9.8 mmole) indiethyl ether (100 ml) was saturated with HCl. After 3 hours the solventwas evaporated under a stream of nitrogen and the residue trituratedwith diethyl ether. Filtration gave a white solid (2.43 g; 94%) m.p.249°-250° C. Found, 59.16; H, 9.83, N, 5.38. C₁₃ H₂₆ ClNO₂ requires C,59.19; H, 9.93; N, 5.31%.

PREPARATION 8 c-4-Amino-t-2-butyl-r-1-cyclohexane carboxylic acid ethylester hydrochloride

(1) cis-7-Butyl-1,4-dioxaspiro[4,5]decane-8-carboxylic acid ethyl ester

cis-2-Butyl-4-oxocyclohexane carboxylic acid ethyl ester (9.0 g; 40mmole), ethylene glycol (2.73 g; 44 mmole) and p-toluenesulphonic acid(100 mg) were refluxed in benzene 80 ml) using a Dean-Stark water trap.After 12 hours the mixture was cooled, diluted with diethyl ether andwashed with saturated aqueous sodium bicarbonate followed by water.Drying over MgSO₄ and evaporation gave a liquid (10.80 g; 100%) whichwas pure enough to use directly. Found: C, 66.71; H, 9.79. C₁₅ H₂₆ O₄requires C, 66.64; H, 9.69%.

(2) trans-7-Butyl-1,4-dioxaspiro[4,5]decane-8-carboxylic acid ethylester

Potassium-tert-butoxide (1.9 g; 17 mmole) was added to a solution of theabove ester (10.75 g; 39.7 mmole) in tert-butanol (90 ml), which hadbeen dried over 3A sieve, and the mixture was refluxed under nitrogenfor 24 hours. The solution was then neutralized with 2N HCl andevaporated to a small volume under reduced pressure. The residue wastaken up in diethyl ether, washed with water, dried over MgSO₄ andevaporated to give a yellow liquid (10.0 g) which was chromatographed onsilica (300 g). Elution with diethyl ether:hexane (2:8) gave therequired trans-isomer as a clear liquid (8.80 g, 82%). Found: C, 66.58;H, 9.67. C₁₅ H₂₆ O₄ requires C, 66.64; H, 9.69%.

(3) trans-2-Butyl-4-oxocyclohexane carboxylic acid ethyl ester

The above ester (8.75 g; 32.4 mmole) in absolute ethanol (70 ml) wasadded to 1N sulphuric acid (50 ml) and the mixture was refluxed for 3hours. Half the solvent was evaporated and the residual suspension wasextracted with diethyl ether. The extract was washed with saturatedaqueous sodium bicarbonate followed by water, dried (MgSO₄) andevaporated to give a clear liquid (6.85 g). Chromatography on silica(500 g) eluting with diethyl ether:hexane (2:8) gave the required ketone(6.05 g; 83%) as a clear liquid. Found: C, 68.73; H, 9.85. C₁₃ H₂₂ O₃requires C, 68.99; H, 9.80%.

(4) trans-2-Butyl-4-methoximinocyclohexane carboxylic acid ethyl ester

The above ketone (6.02 g; 26.6 mmole) methoxylamine hydrochloride (2.89g; 34.6 mmole), sodium acetate (2.84 g; 34.6 mmole) were refluxed inabsolute ethanol (100 ml) for 3 hours. After standing overnight at roomtemperature, most of the solvent was evaporated under reduced pressureand the residue was partitioned between diethyl ether and water. Theether extract was washed with saturated aqueous sodium bicarbonatefollowed by water, dried (MgSO₄) and evaporated to give a clear oil(6.77 g; 100%) which was pure enough to use directly. Found: C, 65.78;H, 9.71; N, 5.43. C₁₄ H₂₅ NO₃ requires C, 65.85; H, 9.87; N, 5.49%.

(5) t-2-Butyl-c-4-(1,1-dimethylethoxycarbonylamino)-1-r-cyclohexanecarboxylic acid ethyl ester

Trifluoroacetic acid (10.2 ml; 0.13 mmole) in dry tetrahydrofuran (20ml) was added dropwise under nitrogen to a stirred suspension of sodiumborohydride (5.0 g; 0.13 mmole) in dry tetrahydrofuran (120 ml). Thetemperature was kept between 10°-20° C. with ice cooling and after 15minutes a solution of the above ester (6.75 g; 26.4 mmole) intetrahydrofuran (20 ml) was added. The temperature rose to 33° C. andbrief cooling was required to return the temperature to 20° C. After 4hours water was carefully added, with ice cooling, followed by diethylether. The organic phase was washed with saturated salt solution, dried(MgSO₄) and evaporated to give crude amine (8.2 g). This product wasdissolved in dry methylene chloride (100 ml) containing N-methylmorpholine (2.67 g; 26.4 mmole), di-tert-butyldicarbonate (11.52 g; 52.8mmole) was added and the solution allowed to stand at room temperaturefor 48 hours. The solvent was evaporated and the residue partitionedbetween diethyl ether and water. The ether extract was washedsuccessively with 0.5N hydrochloric acid, water, saturated aqueoussodium bicarbonate and water. Drying over MgSO₄ and evaporation gave anoil (8.86 g). Chromatography on silica eluting with diethyl ether:hexane(2:8) gave the required cis-isomer as an oil (1.61 g; 19%). Rf 0.35(silica, ether:hexane 3:7). NMR: δ=2.13 (HC--CO.sub. 2 Et), δ=3.88(HC--NH--). Found: C, 65.96; H, 10.14; N, 4.20. C₁₈ H₃₃ NO₄ requires C,66.02; H, 10.16; N, 4.28%. Continued elution then gave the more polartrans-isomer as a white solid (1.53 g; 18%). Recrystallization fromhexane gave a white solid m.p. 78°-9° C. Rf=0.3. NMR: δ=2.1 (HC--CO₂Et), δ=3.49 (HC--NH--). Found: C, 66.08; H, 10.24; N, 4.17. C₁₈ H₃₃ NO₄requires C, 66.02; H, 10.66; N, 4.28%.

(6) c-4-Amino-t-2-butyl-r-1-cyclohexane carboxylic acid ethyl esterhydrochloride

An ice cold solution of the above cis-isomer (1.58 g; 4.83 mmole) indiethyl ether (50 ml) was saturated with HCl. After 3 hours the solventwas evaporated under a stream of nitrogen and the residue was trituratedwith diethyl ether. Filtration gave the required amine salt as a whitesolid (1.17 g; 92%), m.p. 166°-7° C. Found: C, 59.18; H, 10.27; N, 5.19.C₁₃ H₂₆ ClNO₂ requires C, 59.19; H, 9.93; N, 5.31%.

We claim:
 1. A compound having the formula: ##STR160## wherein R³ and R⁴are each independently hydrogen or (C₁ -C₆)alkyl;R⁵ is ##STR161## R⁸ isR⁹ CONH-- or R⁹ SO₂ NH--; R⁹ is (C₁ -C₆)alkyl, (C₃ -C₇)cycloalkyl,naphthyl, phenyl or phenyl substituted by chloro, methyl or methoxy; andR¹¹ is hydrogen, (C₁ -C₆)alkyl or (C₁ -C₆)alkyl substituted by amino orbenzyloxycarbonylamino; or a pharmaceutically acceptable salt thereof.2. A compound of claim 1 wherein R⁴ is H.
 3. A compound of claim 2wherein R⁵ is N² -acetyl-L-lysyl-amino, N² -benzoyl-L-lysyl-amino, N²-naphthoyl-L-lysyl-amino, or N² -methanesulphonyl-L-lysyl-amino.
 4. Acompound of claim 3 wherein R⁵ is N² -acetyl-L-lysyl-amino.
 5. Thecompound of claim 4 which is 2-(N²-acetyl-L-lysyl-aminomethyl)-3-{[(cis-4-carboxy-cyclohexyl)carbamoyl]cyclopentyl}-propanoicacid.
 6. The compound of claim 4 which is 2-(N²-acetyl-L-lysyl-aminomethyl)-3-{-[cis-4-carboxy-cis-3-butyl-cyclohexyl)carbamoyl]cyclopenty}propanoicacid.
 7. The compound of claim 4 which is 2-(N²-acetyl-L-lysyl-aminomethyl)-3-{-[cis-4-carboxy-trans-3-butyl-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid.
 8. A compound of claim 3 wherein R⁵ is N²-methanesulfonyl-L-lysyl-amino.
 9. The compound of claim 8 which is2-(N²-methanesulphonyl-L-lysylaminomethyl-3-{-[cis-4-carboxy-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid.
 10. The compound of claim 8 which is 2-(N²-methanesulphonyl-L-lysylaminomethyl-3-{1-[cis-4-carboxy-cis-3-butylcyclohexyl)carbamoyl]cyclopentyl}propanoicacid.
 11. The compound of claim 8 which is 2-(N²-methanesulphonyl-L-lysylaminomethyl-3-{-[cis-4-carboxy-cis-3-(3-methylbutyl)cyclohexyl)carbamoyl]cyclopentyl}propanoicacid.
 12. A compound of claim 3 wherein R³ is H.
 13. The compound ofclaim 12 which is 2-(N²-benzoyl-L-lysyl-aminomethyl)-3-{-[cis-4-carboxy-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid.
 14. The compound of claim 12 which is 2-(N²-napthoyl-L-lysyl-aminomethyl)-3-{1-[cis-4-carboxy-cyclohexyl)carbamoyl]cyclopentyl}propanoicacid.
 15. A pharmaceutical composition for administration to a mammal inthe treatment of cardiovascular disorders which are mediated by atrialnatriuretic factor comprising a zinc-dependent, neutral endopeptidaseinhibiting amount of a compound of claim 30 together with apharmaceutically acceptable diluent or carrier.
 16. A method forinhibiting zinc-dependent, neutral endopeptidase in the treatment ofcardiovascular disorders which are mediated by atrial natriuretic factorin a mammal which comprises administering to said mammal azinc-dependent, neutral endopeptidase inhibiting amount of a compound ofclaim 30.